The insulin signaling pathway is essential for the regulation of glucose homeostasis, contributes to the regulation of energy substrate metabolism, and is intimately linked to aging and longevity. Because of the prevalence of obesity and type-2 diabetes within industrialized societies, understanding the molecular mechanism of this pathway is an increasing biomedical priority. Insulin like molecules alter system-wide protein turnover, yet the effects on individual proteins is unclear. A change in a protein's turnover can alter the stoichiometry of the active protein and ultimately affect the efficiency of a pathway. Because of the potential functional consequence of protein turnover and the importance of insulin signaling, we propose to study both these processes in the nematode Caenorhabditis elegans using quantitative proteornics. Specific Aim 1 is to develop quantitative stable isotope tracer methodology for both the directed and undirected analysis of protein turnover in C. elegans. Specific Aim 2 will measure the turnover of proteins involved in the insulin/insulin growth factor-1 (IGF-1) signaling pathway of C. elegans in response to the inhibition and overexpression of ligands of the insulin receptor (DAF-2). Specific Aim 3 is to measure the effect of insulin like molecules on the tissue-specific intracellular metabolism of the nematode C. elegans using stable isotope labeling and isotopomer distribution analysis. The successfulcompletion of this proposal will produce methodological frameworkfor studying protein turnover in C. elegans and provide foundation for extending these technologies to higher organisms. Furthermore, the proteome-wide measurement of protein turnover in response to insulin-like molecules will produce an experimental dataset for probing the underlying mechanism of insulin signaling.